Bioproduction of cis‐(1S,2R)‐indandiol,a chiral pharmaceutical intermediate,using a solid–liquid two‐phase partitioning bioreactor for enhanced removal of inhibitors

BACKGROUND: A solid‐liquid two‐phase partitioning bioreactor (TPPB) was used in the biotransformation of indene to cis‐(1S,2R)‐indandiol by Pseudomonas putida 421‐5 (ATCC 55687). Metered substrate feeding in single‐phase operation, or delivery from an immiscible liquid, have previously been employed to regulate the exposure of the biocatalyst to inhibitory concentrations of the substrate. In contrast, the solid‐liquid platform provided in situ substrate addition (ISSA) as well as simultaneous it in situ product removal (ISPR) as a means of overcoming substrate and product toxicity. Three different modes of operation were compared for their effects on the performance of this biotransformation: single‐phase, fed‐batch operation was carried out as a benchmark in 2.75 L aqueous medium, and subsequently with the inclusion of either 700 g liquid silicone oil or 700 g solid polymer beads. RESULTS: Biphasic modes achieved a 3‐fold productivity improvement with respect to single‐phase (30 to 90 mg L^?1 h^?1), and solid‐liquid productivity was similar to liquid‐liquid operation while achieving more extensive removal of inhibitory compounds resulting in a slightly higher product titer (1.29 vs 1.16 g L^?1). The operability of the reactor was improved by the phase stability of the solid polymer beads relative to immiscible organic solvents, preventing emulsion formation and facilitating analytics. CONCLUSION: Solid polymer beads replaced the immiscible liquid auxiliary phase for substrate delivery while performing simultaneous inhibitory molecule sequestration. Copyright © 2011 Society of Chemical Industry     

Biofiltration control of pulping odors – hydrogen sulfide: performance,macrokinetics and coexistence effects of organo‐sulfur species

The work reported here describes the aerobic biodegradation of reduced sulfur compound mixtures in air streams by biofilters. Rates of removal of hydrogen sulfide as a sole substrate and in the presence of organo‐sulfur compounds were determined to see if there were any inhibitory effects of the organo‐sulfur compounds on the rate of hydrogen sulfide removal. Experiments were conducted in three bench‐scale biofilters packed with the mixtures of compost/perlite (4:1), hog fuel/ perlite (4:1), and compost/hog fuel/perlite (2:2:1), respectively. Hydrogen sulfide, the predominant odorous gas produced from kraft pulping processes, was used as the main pollutant (substrate). Other organo‐sulfur species (dimethyl sulfide and dimethyl disulfide), also emitted from kraft pulp mills, were used as competing (secondary) substrates in the waste gas stream. To describe rates of removal a Michaelis–Menten type kinetic equation was modified to incorporate the plug flow behavior of biofilters, and used in evaluating the pseudo‐kinetic parameters, V_max (the maximum removal rate) and K_m (the half saturation concentration), for hydrogen sulfide biodegradation, and the type of macrokinetic competition between hydrogen sulfide and the organo‐sulfur compounds. No significant differences in V _max for the three biofilters were observed. The V _max ranged between 136 and 147 g m⁻³ h ⁻¹, while the K_m varied from 44 to 59 ppmv for the three biofilters. Hydrogen sulfide elimination capacity was not affected by the presence of any of the organo‐sulfur species in all of the three biofilters, confirming earlier results that hydrogen sulfide removal in biofilters is independent of the presence of organo‐sulfur compounds mainly because of its easy biodegradability. © 1999 Society of Chemical Industry     

Production of 1‐Octanol from n‐Octane by Pseudomonas putida KT2440

A two‐phase biotransformation process for selective hydroxylation of n‐octane to 1‐octanol via Pseudomonas putida KT2440 harboring heterologously expressed P450 monooxygenase from Mycobacterium marinum is presented. Maximum cell‐specific conversion rates of 12.7 mg_octanol g_CDWh^–1 were observed not only in shaking flasks but also in 3.7‐L‐bioreactor studies. The bioreactor experiments were performed avoiding explosive gas mixtures by lowering volumetric power input, aeration rates and substrate concentrations. Based on a stoichiometric network of P. putida KT2440 topological studies were carried out. As a conclusion, potential limitations of NAD(P)H and/or ATP supply at production conditions can be excluded. Hence, the great potential of the host for further increasing conversion is outlined.     

An Ab Initio Theoretical Study of 2,4,6‐Trinitro‐1,3,5‐Triazine, 3,6‐Dinitro‐1,2,4,5‐Tetrazine,and 2,5,8‐Trinitro‐Tri‐s‐Triazine

In order to evaluate 2,4,6‐trinitro‐1,3,5‐triazine (TNTAz), 3,6‐dinitro‐1,2,4,5‐tetrazine (DNTAz), and 2,5,8‐trinitro‐tri‐s‐triazine (TNTsTAz), the geometries of these compounds have been fully optimized employing the B3LYP density functional method and the AUG‐cc‐pVDZ basis set. The accurate gas phase enthalpies of formation have been obtained by using the atomization procedure and designing isodesmic reactions in which the parent rings are not destroyed. Based on B3LYP/AUG‐cc‐pVDZ calculated geometries and natural charges, the crystal structures have been predicted using the Karfunkel–Gdanitz method. Computed results show that there exists extended conjugation over the parent rings of these compounds. More energy content is reserved in DNTAz than in both TNTAz and TNTsTAz. The title compounds are much more sensitive than 1,3,5‐trinitrobenzene. The calculated detonation velocity of DNTAz reaches 9.73–9.88 km s⁻¹, being larger than those of CL‐20 and TNTAz. TNTsTAz has no advantage over the widely used energetic compounds such as RDX and HMX.     

Design,Synthesis, in vitro MAO‐B Inhibitory Evaluation,and Computational Studies of Some 6‐Nitrobenzothiazole‐Derived Semicarbazones

Monoamine oxidase B (MAO‐B) is an important drug target for the treatment of neurological disorders. A series of 6‐nitrobenzothiazole‐derived semicarbazones were designed, synthesized, and evaluated as inhibitors of the rat brain MAO‐B isoenzyme. Most of the compounds were found to be potent inhibitors of MAO‐B, with IC₅₀ values in the nanomolar to micromolar range. Molecular docking studies were performed with AutoDock 4.2 to deduce the affinity and binding mode of these inhibitors toward the MAO‐B active site. The free energies of binding (ΔG) and inhibition constants (K_i) of the docked compounds were calculated by the Lamarckian genetic algorithm (LGA) of AutoDock 4.2. Good correlations between the calculated and experimental results were obtained. 1‐[(4‐Chlorophenyl)(phenyl)methylene]‐4‐(6‐nitrobenzothiazol‐2‐yl)semicarbazide emerged as the lead MAO‐B inhibitor, with top ranking in both the experimental MAO‐B assay (IC₅₀: 0.004±0.001 μM ) and in computational docking studies (K_i: 1.08 μM ). Binding mode analysis of potent inhibitors suggests that these compounds are well accommodated by the MAO‐B active site through stable hydrophobic and hydrogen bonding interactions. Interestingly, the 6‐nitrobenzothiazole moiety is stabilized in the substrate cavity with the aryl or diaryl residues extending up into the entrance cavity of the active site. According to our results, docking experiments could be an interesting approach for predicting the activity and binding interactions of this class of semicarbazones against MAO‐B. Thus, a binding site model consisting of three essential pharmacophoric features is proposed, and this can be used for the design of future MAO‐B inhibitors.     

Plasma‐polymerized hexamethyldisiloxane films characterized by variable‐energy positron lifetime spectroscopy

Nanometer‐size holes in plasma‐polymerized thin films were characterized by variable‐energy positron lifetime spectroscopy for the first time. Hexamethyldisiloxane (HMDSiO) was plasma‐polymerized at different discharge powers (30–105 W) and monomer pressures (1.0–4.9 Pa). The positron lifetime spectra of deposited films were collected at positron energies of 1 and 5 keV. All films showed a well‐defined long‐lived component due to pick‐off annihilation of ortho‐positronium (o‐Ps). The o‐Ps lifetime τ₃, reflecting the average size of free‐volume holes in the film, increased with an increasing ratio of plasma discharge power, W, and monomer flow rate, F. Based on the empirical relationship between the o‐Ps lifetime and the cavity radius, hole volumes were estimated to be 0.19–0.36 nm³. We also found that the o‐Ps intensity, I₃, depends strongly on the same parameter, W/F. Comparison with infrared (IR) absorption spectroscopy data showed that Ps formation is suppressed in films with fewer organic bonds and higher disorder, i.e., those increasingly inorganic in nature. © 1999 John Wiley & Sons, Inc. J Appl Polym Sci 74: 2522–2528, 1999     

Assessment of aged biodegradable polymer‐coated nano‐zero‐valent iron for degradation of hexahydro‐1,3,5‐trinitro‐1,3,5‐triazine (RDX)

BACKGROUND: This study reports on the effects of aging on suspension behavior of biodegradable polymer‐coated nano‐zero‐valent iron (nZVI) and its degradation rates of hexahydro‐1,3,5‐trinitro‐1,3,5‐triazine (RDX) under reductive conditions. The polymers investigated included guar gum, potato starch, alginic acid (AA), and carboxymethyl cellulose (CMC). Polymer coating was used to mitigate nZVI delivery hindrance for in situ treatment of RDX‐contaminated groundwater. RESULTS: The RDX degradation rates by bare nZVI and starch‐coated nZVI suspensions were least affected by aging although these suspensions exhibited the least favorable dispersion behavior. CMC, AA, and guar gum coating improved nZVI rates of degradation of RDX but these rates decreased upon aging. The best suspension stability upon aging was achieved by CMC and AA. Guar gum with loadings rates one order of magnitude lower than that of CMC and AA achieved good iron stabilization but significantly higher RDX degradation rates. CONCLUSION: It is demonstrated that both migration and reactivity of polymer‐stabilized nZVI should be explicitly evaluated over a long period before application in the field. Guar gum coated nZVI appeared best suited for in situ application because it maintained good suspension stability, with RDX degradation rates least affected by aging compared with the other polymers tested. © 2012 Society of Chemical Industry     

One‐Pot Cascade Reactions using Fructose‐6‐phosphate Aldolase: Efficient Synthesis of D‐Arabinose 5‐Phosphate,D‐Fructose 6‐Phosphate and Analogues

One‐pot multienzymatic reactions have been performed for the synthesis of 1‐deoxy‐D ‐fructose 6‐phosphate, 1,2‐dideoxy‐D ‐arabino‐hept‐3‐ulose 7‐phosphate, D ‐fructose 6‐phosphate and D ‐arabinose 5‐phosphate. The whole synthetic strategy is based on an aldol addition reaction catalysed by fructose‐6‐phosphate aldolase (FSA) as a key step of a three or four enzymes‐catalysed cascade reaction. The four known donors for FSA – dihydroxyacetone (DHA), hydroxyacetone (HA), 1‐hydroxy‐2‐butanone (HB) and glycolaldehyde (GA) – were used with D ‐glyceraldehyde 3‐phosphate as acceptor substrate. The target phosphorylated sugars were obtained in good to excellent yields and high purity.     

Anticancer Ruthenium(III) Complex KP1019 Interferes with ATP‐Dependent Ca2+ Translocation by Sarco‐Endoplasmic Reticulum Ca2+‐ATPase (SERCA)

Sarco‐endoplasmic reticulum Ca²⁺‐ATPase (SERCA), a P‐type ATPase that sustains Ca²⁺ transport and plays a major role in intracellular Ca²⁺ homeostasis, represents a therapeutic target for cancer therapy. Here, we investigated whether ruthenium‐based anticancer drugs, namely KP1019 (indazolium [trans‐tetrachlorobis(1H‐indazole)ruthenate(III)]), NAMI‐A (imidazolium [trans‐tetrachloro(1H‐imidazole)(S‐dimethylsulfoxide)ruthenate(III)]) and RAPTA‐C ([Ru(η⁶‐p‐cymene)dichloro(1,3,5‐triaza‐7‐phosphaadamantane)]), and cisplatin (cis‐diammineplatinum(II) dichloride) might act as inhibitors of SERCA. Charge displacement by SERCA adsorbed on a solid‐supported membrane was measured after ATP or Ca²⁺ concentration jumps. Our results show that KP1019, in contrast to the other metal compounds, is able to interfere with ATP‐dependent translocation of Ca²⁺ ions. An IC₅₀ value of 1 μM was determined for inhibition of calcium translocation by KP1019. Conversely, it appears that KP1019 does not significantly affect Ca²⁺ binding to the ATPase from the cytoplasmic side. Inhibition of SERCA at pharmacologically relevant concentrations may represent a crucial aspect in the overall pharmacological and toxicological profile of KP1019.     

Lecithin‐enhanced biotransformation of cholesterol to androsta‐1,4‐diene‐3,17‐dione and androsta‐4‐ene‐3,17‐dione

A biotransformation process using Mycobacterium sp was studied for androsta‐1, 4‐diene‐3,17‐dione (ADD) and androsta‐4‐ene‐3,17‐dione (AD) production from cholesterol. Cholesterol has a poor solubility in water (～1.8 mg dm^?3 at 25 °C), which makes it difficult to use as the substrate for biotransformation. Lecithin is a mixture of phospholipids of phosphatidylcholine (PC) and phosphatidylethanolamine (PE), which behave like surfactants and can form planar bi‐layer structures in an aqueous medium. Therefore, a small amount of lecithin (<1 g dm^?3) can be used to form stable colloids with cholesterol at a relatively high concentration (20 g dm^?3) in water. In this work, an energy density of 1000 J cm^?3 from sonication was provided to overcome the self‐association of cholesterol and to generate a stable lecithin–cholesterol suspension that could be used for enhanced biotransformation. The lecithin–cholesterol suspension was stable and could withstand typical autoclaving conditions (121 °C, 15 psig, 20 min). In contrast to conventional surfactants, such as Tween 80, that are commonly used to help solubilize cholesterol, lecithin did not change the surface tension of the aqueous solution nor cause any significant foaming problem. Lecithin was also biocompatible and showed no adverse effect on cell growth. Compared with the medium with Tween 80 as the cholesterol‐solubilizing agent, lecithin greatly improved the biotransformation process in regard to its final product yield (～59% w/w), productivity (0.127–0.346 g dm^?3 day^?1), ADD/AD ratio (6.7–8), as well as the long‐term process stability. Cells can be reused in repeated batch fermentations for up to seven consecutive batches, but then lose their bioactivity due to aging problems, possibly caused by product inhibition and nutrient depletion. © 2002 Society of Chemical Industry     

Improved modelling of the unsteady‐state behaviour of an immobilized‐cell,fluidized‐bed bioreactor for phenol biodegradation

The dynamic response of an immobilized‐cell, fluidized‐bed reactor (ICFBR) to step changes in phenol loading was investigated at 10°C for a pure culture of Pseudomonas putida Q5, a psychrotrophic bacterium. A novel dynamic model was developed and tested to simulate the response of all four key process variables: the bulk phenol concentration, the suspended biomass concentration, the concentration profile of the substrate in the biofilm and the biofilm thickness. Accurate model predictions required the use of kinetics, determined using cells which were not acclimated to the post‐shock reactor conditions (‘unacclimated’ cells) and the implementation of a specific‐growth‐rate suppression factor to account for the unbalanced growth situations experienced during the transient response periods.     

Selective Extraction of N‐Heterocyclic Precursors of 1,3,5,7‐Tetranitro‐1,3,5,7‐tetraazacyclooctane (HMX) Using Molecularly Imprinted Polymers

N‐heterocyclic compounds are key nitration precursors for some high energy density explosives such as 1,3,5,7‐tetranitro‐1,3,5,7‐tetraazacyclooctane (HMX). Nitration of 1,3,5,7‐tetraacetyl‐1,3,5,7‐tetraazacyclooctane (TAT) yields HMX in high yields and purity. However, the analogue 1,3,5‐triacetyl‐1,3,5‐triazacyclohexane (TRAT) is easily co‐produced via the condensation of acetonitrile and 1,3,5‐trioxan. To selectively extract TAT from a mixture of TAT and TRAT, the molecular imprinting technology (MIT) was developed in this study. The capacity of the dry polymer is 16 mg g⁻¹ and the recovery surpasses 75 %.     

Nitrification by immobilized cells in a micro‐ecological life support system using packed‐bed bioreactors: an engineering study

The nitrifying component of a micro‐ecological life support system alternative (MELISSA) based on microorganisms and higher plants was studied. The MELISSA system consists of an interconnected loop of bioreactors to allow the recycling of the organic wastes generated in a closed environment. Conversion of ammonia into nitrates in such a system was improved by selection of microorganisms, immobilization techniques, reactor type and operation conditions. An axenic mixed culture of Nitrosomonas europaea and Nitrobacter winogradskyi, immobilized by surface attachment on polystyrene beads, was used for nitrification in packed‐bed reactors at both bench and pilot scale. Hydrodynamics, mass transfer and nitrification capacity of the reactors were analysed. Mixing and mass transfer rate were enhanced by recirculation of the liquid phase and aeration flow‐rate, achieving a liquid flow distribution close to a well‐mixed tank and without oxygen limitation for standard operational conditions of the nitrifying unit. Ammonium conversion ranged from 95 to 100% when the oxygen concentration was maintained above 80% of saturation. The maximum surface removal rates were measured as 1.91 gN‐NH₄⁺ m^?2 day^?1 at pilot scale and 1.77 gN‐NH₄⁺ m^?2 day^?1 at bench scale. Successful scale‐up of a packed‐bed bioreactor has been carried out. Good stability and reproducibility were observed for more than 400 days. Copyright © 2004 Society of Chemical Industry     

Degradation of hexahydro‐1,3,5‐trinitro‐1,3, 5‐triazine (RDX) by anaerobic mesophilic granular sludge from a UASB reactor

BACKGROUND: This study investigated the performance of anaerobic mesophilic granular sludge for the degradation of hexahydro‐1,3,5‐trinitro‐1,3,5‐triazine (RDX). Batch tests were conducted to investigate the effects of different supplements on the RDX degradation ability of anaerobic granular sludge, as well as the contributions of both physicochemical and biological processes involved in RDX removal from aqueous solution. RESULTS: Anaerobic granular sludge exhibited good performance in treating RDX as the sole substrate. Biodegradation was the main mechanism responsible for RDX removal. Ammonium had no significant promoting effect on the degradation process. The presence of glucose was found to enhance the degradation of RDX by anaerobic granular sludge, while the addition of sulfate and nitrate had adverse effects on the reductive transformation of RDX. CONCLUSIONS: Anaerobic granular sludge is capable of removing RDX from aqueous solution with high efficiency. This study showed good prospects for high‐rate anaerobic processes in the treatment of munition wastewater. The results can be used for the design and optimization of high rate anaerobic systems for the elimination of RDX. Copyright © 2010 Society of Chemical Industry     

Biotransformation of (−)‐bornyl acetate using submerged cultures of Collybia velutipes,Trametes hirsuta and Ganoderma applanatum

The biotransformation of (?)‐bornyl acetate by three Basidiomycetes, Collybia velutipes, Trametes hirsuta and Ganoderma applanatum was monitored for 7 days. The observed reactions were regio‐ and stereo‐selective hydroxylation, acetate hydrolysis and oxidation of alcohols to carbonyl compounds. Nine cyclic compounds, three of which (6‐exo‐hydroxybornyl acetate, 8‐hydroxy‐5‐exo‐hydroxybornyl acetate, 9‐hydroxy‐5‐exo‐hydroxybornyl acetate), not previously described, were isolated from the fermentation broth and therefore were identified. Product types and concentrations varied with strain and incubation time. The toxicity of the substrate was assessed from the variation of fungal biomass in the course of incubation. Copyright © 2005 Society of Chemical Industry     

Microbial conversion of androst‐1,4‐dien‐3,17‐dione by Mucor racemosus to hydroxysteroid‐1,4‐dien‐3‐one derivatives

BACKGROUND: A large number of bacterial, fungal and microalgal species are able to bio‐transform steroid compounds. Among them, fungi from the Mucor genus have been shown to mediate hydroxylation, oxidation, and desaturation by the double bond formation and epoxidation of various steroid substances. Mucor racemocus has not been studied for its ability to modify androst‐1,4‐dien‐3,17‐dione, a pharmaceutically important steroid precursor. RESULTS: The filamentous fungus M. racemosus was applied for bioconversion of androst‐1,4‐dien‐3,17‐dione (ADD, I ) in a 5‐day fermentation. Microbial metabolites were purified chromatographically and identified on the basis of their spectral data as 17β‐hydroxyandrost‐1,4‐dien‐3‐one ( II ), 14α‐hydroxyandrost‐1,4‐dien‐3,17‐dione ( III ), 15α‐hydroxyandrost‐1,4‐dien‐3,17‐dione ( IV ), 15α,17β‐dihydroxyandrost‐1,4‐dien‐3‐one ( V ), 14α,17β‐dihydroxyandrost‐1,4‐dien‐3‐one ( VI ), and 6β,17β‐dihydroxyandrost‐1,4‐dien‐3‐one ( VII ). CONCLUSION: Observed modifications included hydroxylation at C‐6β, C‐14α, C‐15α positions and 17‐carbonyl reduction. The best fermentation conditions for production of hydroxysteroid‐1,4‐dien‐3‐one derivatives were found to be 25 °C at 150 rpm for 5 days with a substrate concentration of 0.5 g L^?1. Copyright © 2009 Society of Chemical Industry     

Biodegradation kinetics of trans‐4‐methyl‐1‐cyclohexane carboxylic acid in continuously stirred tank and immobilized cell bioreactors

BACKGROUND: Naphthenic acids are carboxylic acid compounds of oil sands wastewaters that contribute to aquatic toxicity. Biodegradation kinetics of an individual naphthenic acid compound in two types of continuous‐flow bioreactors were investigated as a means of improving remediation strategies for these compounds. RESULTS: This study evaluates the kinetics of biodegradation of trans‐4‐methy‐1‐cyclohexane carboxylic acid (trans‐4MCHCA) using two bioreactor systems and a microbial culture developed in previous work. Using a feed concentration of 500 mg L^?1 the biodegradation rate of trans‐4MCHCA in the immobilized cell bioreactor was almost two orders of magnitude higher than that in a continuously stirred tank bioreactor. The maximum reaction rates of 230 mg (L d)^?1 at a residence time of 1.6 d (40 h) and 22 000 mg (L d)^?1 at a residence time of 2.6 h were observed in the continuously stirred tank and immobilized cell bioreactors, respectively. In a second immobilized cell system operating with a feed concentration of 250 mg L^?1, a comparable maximum reaction rate (21 800 mg (L d)^?1) was achieved at a residence time of 1.0 h. CONCLUSION: The use of immobilized cell bioreactors can enhance the biodegradation rate of naphthenic acid compounds by two orders of magnitude. Further, biodegradation greatly reduces the toxicity of the effluent wastewater. Copyright © 2009 Society of Chemical Industry     

A Mutant D‐Fructose‐6‐Phosphate Aldolase (Ala129Ser) with Improved Affinity towards Dihydroxyacetone for the Synthesis of Polyhydroxylated Compounds

A mutant of D ‐fructose‐6‐phosphate aldolase (FSA) of Escherichia coli, FSA A129S, with improved catalytic efficiency towards dihydroxyacetone (DHA), the donor substrate in aldol addition reactions, was explored for synthetic applications. The k_cat/K_M value for DHA was 17‐fold higher with FSA A129S than that with FSA wild type (FSA wt). On the other hand, for hydroxyacetone as donor substrate FSA A129S was found to be 3.5‐fold less efficient than FSA wt. Furthermore, FSA A129S also accepted glycolaldehyde (GA) as donor substrate with 3.3‐fold lower affinity than FSA wt. This differential selectivity of both FSA wt and FSA A129S for GA makes them complementary biocatalysts allowing a control over donor and acceptor roles, which is particularly useful in carboligation multi‐step cascade synthesis of polyhydroxylated complex compounds. Production of the mutant protein was also improved for its convenient use in synthesis. Several carbohydrates and nitrocyclitols were efficiently prepared, demonstrating the versatile potential of FSA A129S as biocatalyst in organic synthesis.     

Physiological comparison of Pseudomonas putida between two growth phases during cometabolism of 4‐chlorophenol in presence of phenol and glutamate: a proteomics approach

BACKGROUND: In cometabolic transformation of 4‐chlorophenol (4‐cp) in the presence of phenol and sodium glutamate (SG), a new biphasic growth pattern has been reported. This study investigates the physiological changes of Pseudomonas putida P8 during the biphasic growth by means of 2‐dimensional gel electrophoresis (2‐DE) and MALDI‐TOF analyses. RESULTS: A total of 49 protein spots were selected and identified in the 2‐DE gels from P. putida P8 grown on a mixed substrate containing 200 mg L^?1 of phenol, 200 mg L^?1 of 4‐cp and 1000 mg L^?1 SG. Among them, 16 protein spots were found differentially expressed in the two exponential growth phases during the biphasic growth, including six catabolic enzymes (DmpC, DmpD, DmpE, DmpF, DmpG and AspA) for substrate utilization. The expression of other proteins involved in detoxification and stress responses, carbohydrate and energy metabolism, and environmental information processing as well as a multifunctional xenobiotic reductase (XenA) was quantitatively analyzed and discussed. CONCLUSION: The expression levels of the identified catabolic enzymes during growth in the two growth phases correlated well with the substrate utilization patterns observed in previous kinetics studies. Furthermore, the results show that cells growing on a mixture of aromatic substrates undergo significant physiological changes. Copyright © 2009 Society of Chemical Industry     

4‐Hydroxy‐3‐methyl‐6‐(1‐methyl‐2‐oxoalkyl)pyran‐2‐one Synthesis by a Type III Polyketide Synthase from Rhodospirillum centenum

The purple photosynthetic bacterium Rhodospirillum centenum has a putative type III polyketide synthase gene (rpsA). Although rpsA was known to be transcribed during the formation of dormant cells, the reaction catalyzed by RpsA was unknown. Thus we examined the RpsA reaction in vitro, using various fatty acyl‐CoAs with even numbers of carbons as starter substrates. RpsA produced tetraketide pyranones as major compounds from one C_10–14 fatty acyl‐CoA unit, one malonyl‐CoA unit and two methylmalonyl‐CoA units. We identified these products as 4‐hydroxy‐3‐methyl‐6‐(1‐methyl‐2‐oxoalkyl)pyran‐2‐ones by NMR analysis. RpsA is the first bacterial type III PKS that prefers to incorporate two molecules of methylmalonyl‐CoA as the extender substrate. In addition, in vitro reactions with ¹³C‐labeled malonyl‐CoA revealed that RpsA produced tetraketide 6‐alkyl‐4‐hydroxy‐1,5‐dimethyl‐2‐oxocyclohexa‐3,5‐diene‐1‐carboxylic acids from C_14–20 fatty acyl‐CoAs. This class of compounds is likely synthesized through aldol condensation induced by methine proton abstraction. No type III polyketide synthase that catalyzes this reaction has been reported so far. These two unusual features of RpsA extend the catalytic functions of the type III polyketide synthase family.